Recombinase polymerase amplification

ABSTRACT

This disclosure describe three related novel methods for Recombinase-Polymerase Amplification (RPA) of a target DNA that exploit the properties of the bacterial RecA and related proteins, to invade double-stranded DNA with single stranded homologous DNA permitting sequence specific priming of DNA polymerase reactions. The disclosed methods has the advantage of not requiring thermocycling or thermophilic enzymes. Further, the improved processivity of the disclosed methods allow amplification of DNA up to hundreds of megabases in length.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/192,806 filed on Jul. 28, 2011, which is a continuation of U.S.application Ser. No. 12/799,786 filed on Apr. 30, 2010, which is acontinuation of U.S. application Ser. No. 12/322,354 filed on Jan. 30,2009, which is a continuation of U.S. application Ser. No. 11/893,113filed on Aug. 13, 2007, now U.S. Pat. No. 7,485,428, which is acontinuation of U.S. application Ser. No. 10/371,641 filed on Feb. 21,2003, now U.S. Pat. No. 7,270,981, which claims the benefit of priorityto U.S. Application Ser. No. 60/358,563 filed Feb. 21, 2002. Thedisclosures of the above applications are incorporated herein in theirentireties.

BACKGROUND

Throughout this specification, various patents, published patentapplications and scientific references are cited to describe the stateand content of the art. Those disclosures, in their entireties, arehereby incorporated into the present specification by reference.

The ability to amplify DNA lies at the heart of modern biological andmedical research. This is because most molecular biology techniques relyon samples containing many identical molecules to increase thesensitivity of an assay or to prepare enough material for furtherprocessing. Among the various nucleic acid amplification techniques,polymerase chain reaction (PCR) is the most common because of itssensitivity and efficiency at amplifying short nucleic acid sequences.

While PCR is of great utility, it is also limited in a number of ways.The first limitation of PCR is that it relies on multiple cycles ofthermal melting (denaturing) at high temperatures followed byhybridization and elongation at a reduced temperature. To maximizeefficiency and to minimize noise, complex temperature control ofmultiple reactions is required. This necessitates the use of athermocycler controllable rapid heating/cooling block made with exoticmaterial (e.g., gold plated silver blocks), or a robotic mechanism tomove samples between temperature-controlled zones. Because of thehigh-temperature required to melt DNA in physiological salt conditions,PCR technology requires either the addition of fresh polymerase percycle or the use of thermostable polymerases. The approach of addingfresh polymerase has not been automated and is thus labor intensive andprone to errors (e.g., contamination, dropped tubes, labeling errors).Furthermore, the need to add enzymes and to mix each reactionindividually presents serious drawbacks that have limited adaptation ofenzyme-addition PCR methods to the small scale.

Compared to methods involving the addition of fresh polymerase, the useof thermostable polymerases in PCR is the most widely practiced. Thisapproach suffers from the fact that thermostable polymerases are foundin a limited number of organisms, and the replication mechanisms used bythermophilic organisms are poorly understood. The available repertoireof thermostable polymerases is limited to single polypeptide polymeraseenzymes involved in DNA repair, and/or lagging strand synthesis. DNArepair and/or lagging strand polymerases are poor choices for DNAamplification because they exhibit poor processivity (distributivesynthesis). In part as a consequence of using repair and/or laggingstrand polymerases (e.g. Taq, Pfu, Vent polymerases), and due to theformation of inhibitory secondary or tertiary nucleic acid structuresfollowing thermal melting, current PCR protocols do not readily amplifysequences longer than several thousands of base pairs. Reliablesynthesis (and amplification) of longer templates will rely onpolymerases and auxiliary enzymatic complexes collectively exhibitingmuch higher levels of processivity, strand displacement, and secondarystructure resolution, as well as limiting the formation of inhibitoryhigher order nucleic acid structures which may form on coolingheat-denatured DNA.

A second limitation of PCR is that it relies on solution hybridizationbetween oligonucleotides (PCR primers) and denatured template DNA (i.e.,the DNA to be amplified) in an aqueous environment. To be effective, PCRreactions are performed in a short time because the thermostablepolymerases have a rapidly declining activity at PCR temperatures.Further, for effective hybridization in a short time, a feature criticalto rapid turnaround, it is necessary to perform PCR in an environmentwith high concentrations of oligonucleotides. The high oligonucleotideconcentration also ensures rapid interaction of target sequences withthe oligonucleotides in competition with the heat-denaturedcomplementary strand still present in solution. High oligonucleotideprimer concentrations can cause problems, particularly when the copynumber of the target sequence is low and present in a complex mixture ofDNA molecules. This would be the case, for example, in a PCR of a genometo determine the genetic polymorphism in one locus.

One problem with using high oligonucleotide concentrations is that itenhances the degree of false priming at only partly matched sequences inthe complex DNA mixture. False priming refers to the hybridization of aprimer to a template DNA in PCR even when the primer sequence is notcompletely complementary to the template nucleic acid, which can lead tonon-specific amplification of nucleic acids. Noise, due to falsepriming, increases with the oligonucleotide concentration and thecomplexity of total starting DNA. In addition, the possibility of falsepriming increases as the copy number of target sequences decreases.Where the conditions for false priming are favorable (i.e., higholigonucleotide concentration, high complexity, low copy number), errantamplified sequences can become a dominant reaction product. Consequentlyit can be difficult to identify conditions, and oligonucleotides, forclean amplification of target sequences from a sample DNA without anexcess of false priming background. Thus a further disadvantage of usingPCR is the limited success at cleanly amplifying rare target DNAs fromcomplex sequences mixtures.

One solution to the problems of specificity and template-melting problemincurred by PCR is to employ methods that rely on the biologicalproperties of the bacterial RecA protein, or its prokaryotic andeukaryotic relatives. These proteins coat single-stranded DNA (ssDNA) toform filaments, which then scan double-stranded DNA (dsDNA) for regionsof sequence homology. When homologous sequences are located, thenucleoprotein filament strand invades the dsDNA creating a short hybridand a displaced strand bubble known as a D-loop. The free 3′-end of thefilament strand in the D-loop can be extended by DNA polymerases tosynthesize a new complementary strand. The complementary stranddisplaces the originally paired strand as it elongates. By utilizingpairs of oligonucleotides in a manner similar to that used in PCR itshould be possible to amplify target DNA sequences in an analogousfashion but without any requirement for thermal melting (thermocycling).This has the advantage both of allowing the use of heat labilepolymerases previously unusable in PCR, and increasing the fidelity andsensitivity by template scanning and strand invasion instead ofhybridization.

Although the use of RecA and its homologues for in vitro amplificationof nucleic acids has been previously described (U.S. Pat. No. 5,223,414to Zarling et al., referred to herein as “Zarling”), the method andresults are limited. Zarling's method have critical failings, which havelimited its ability to achieve exponential amplification ofdouble-stranded DNA. The failure of the Zarling method to achieveexponential amplification may be due to its specification for the use ofATPγS rather than ATP. The Zarling method urges the use of ATPγS,instead of ATP, in the assembly of RecA nucleoprotein filaments becauseit results in a more stable RecA/ssDNA filament structure. Normally,filaments are assembled in a 5′ to 3′ direction and will spontaneouslydisassemble in the same 5′ to 3′ direction as RecA hydrolyzes ATP. Thisprocess is dynamic in that assembly and disassembly occurs at the sametime and the amount of assembled filaments is at equilibrium. If thenon-hydrolyzable ATP analog, ATPγS, is used, hydrolysis of the ATPγS andthe 5′ to 3′ disassembly of the filaments are inhibited. The greatstability of RecA/ATPγS filaments, both before and after strandexchange, while helpful in the method of targeting (i.e., the Zarlingmethod) is detrimental and unpractical for DNA amplification.

In the Zarling method, RecA protein involved in strand invasion willremain associated with the double-stranded portion of the exchangedmaterial after strand exchange. This interaction occurs because thenewly formed duplex is bound in the high-affinity site of RecA. Thedisplaced strand occupies a different low-affinity site, unless it isbound to another single-stranded DNA binding protein (SSB), such as E.coli SSB. If ATP had been utilized to generate the exchange structure,spontaneous 5′ to 3′ disassembly might occur, although the exchangecomplex can be quite stable and may require additional factors tostimulate ATP-dependent disassembly. Regardless of whether spontaneousor stimulated, in the presence of ATPγS, 5′ to 3′ disassembly of theRecA filament is inhibited (Paulus, B. F. and Bryant, F. R. (1997).Biochemistry 36, 7832-8; Rosselli, W. and Stasiak, A. (1990). J Mol Biol216, 335-52; Shan, Q. et al., (1997). J Mol Biol 265, 519-40). TheseRecA/dsDNA complexes are precisely the sites targeted by the RecA/ssDNAprimer complexes used to initiate subsequent rounds of invasion andsynthesis. With the RecA bound, the dsDNAs can no longer be invaded byRecA/ssDNA primer complexes and are therefore not amplifiable from thispoint. Further synthesis from these templates might occur if initiatedat the other end of the template, which is free of RecA, and this mighteventually lead to displacement of the bound RecA. It is not clear,however, whether many polymerases can displace RecA in this manner.Moreover, the initiation site for that synthetic round will now be‘blocked’ instead. In such a situation, amplification is only linearwith time, and will predominately generate single-stranded DNAamplification products. Thus, the described Zarling method, at best, islikely to generate little more than small quantities of ssDNA copiesfrom each template. In addition, the linear amplification given by theZarling method will only occur in the presence of SSB, since thedisplaced strand will continue to bind to the second interaction site onRecA, and single-stranded DNA will not be released (Mazin, A. V. andKowalczykowski, S. C. (1998). Embo J 17, 1161-8). This probably explainswhy the Zarling method observed additional faster-migrating fragmentswhen they included SSB. These additional fragments were most likelydisplaced single-stranded fragments. Hence, in the Zarling method onlylinear amplification of single-stranded DNA will occur. There is,therefore, a need in the art for an improved recombinase-dependent DNAamplification method.

This invention utilizes two new amplification strategies that avoid anyrequirement for thermal melting of DNA or thermostable components. Thesestrategies also overcome the inefficiencies of the Zarling method. Aswith the Zarling strategy, these methods rely on the biologicalproperties of the bacterial RecA protein, or its prokaryotic andeukaryotic relatives. However, in contrast to the Zarling method, thesemethods are devised to achieve exponential amplification of dsDNA. Theyachieve this by permitting rapid regeneration of targetable sequences inthe target nucleic acid in the presence of dynamic RecA/DNA filaments.Furthermore one of the methods obviates any requirement for phasedreplication initiation from both ends of the target nucleic acid bycoupling leading and lagging strand synthesis to simultaneously generate2 double-stranded products.

BRIEF DESCRIPTION OF THE INVENTION

The invention provides a method of DNA amplification, termed RPA, whichcomprises the following steps. First, a recombinase agent is contactedwith a first and a second nucleic acid primer to form a first and asecond nucleoprotein primer. Second, the first and second nucleoproteinprimers are contacted to a double stranded target sequence to form afirst double stranded structure at a first portion of said first strandand form a double stranded structure at a second portion of said secondstrand so the 3′ ends of said first nucleic acid primer and said secondnucleic acid primer are oriented towards each other on a given templateDNA molecule. Third, the 3′ end of said first and second nucleoproteinprimers are extended by DNA polymerases to generate first and seconddouble stranded nucleic acids, and first and second displaced strands ofnucleic acid. Finally, the second and third steps are repeated until adesired degree of amplification is reached.

The invention also provides for a method of nested RPAs. In a nestedRPA, a first region of nucleic acid is amplified by RPA to form a firstamplified region. Then a second region of nucleic acid that iscompletely within the first amplified region is amplified using RPA toform a second amplified region. This process may be repeated as often asnecessary. For example, a third region of nucleic acid, which iscompletely within the second region, may be amplified from the secondamplified region by RPA. In addition to the one, two and three rounds ofRPA discussed above, the invention contemplates at least 4, andpreferably at least 5 rounds of nested RPAs also.

The invention also provides for methods of detecting a genotype usingRPA. This method is useful for genotyping, for detecting a normal ordiseased condition, a predisposition or a lack of a disposition for adiseased condition. Further, RPA can be used for detecting the presenceof a genome, such as for example, a genome of a pathogen. In this use,the method is useful for diagnosis and detection.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts a schematic representation of RecA/Primer Loading.

FIGS. 2A-2B depicts a schematic of succeeding steps, shown in panels (a)and (b) of Leading Strand Recombinase Polymerase Amplification (lsRPA).

FIGS. 3A-3D depicts a schematic of succeeding steps, shown in panels (a)(b) (c) and (d), of Leading and Lagging Strand Recombinase PolymeraseAmplification.

FIG. 4 depicts an example of nested primers chosen for nested RPA.

FIG. 5 depicts examples of suitable double stranded template nucleicacids.

FIGS. 6A-6B depicts in panels (a) and (b) the various orientations ofthe RPA primer pairs in hybridization with the target nucleic acid.

FIGS. 7A-7C panels (a), (b) and (c) depicts a schematic representationof an RPA reaction in progress.

FIGS. 8A-8C depicts (a) examples of double stranded primers (b) doublestranded primers after elongation and after annealing of the secondmember of a primer pair (c) after the elongation of the second member ofa primer pair with the non-invading strand displaced.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides for Recombinase-Polymerase Amplification(RPA)—a method for the amplification of target nucleic acid polymers.One benefit of RPA is that it may be performed without the need forthermal melting of double-stranded templates therefore, the need forexpensive thermocyclers is also eliminated. The present inventiondescribes two related strategies by which RPA can be configured topermit exponential amplification of target nucleic acid polymers.

Leading Strand Recombinase-Polymerase Amplification (lsRPA)

In leading strand Recombinase-polymerase Amplification (lsRPA)single-stranded, or partially single-stranded, nucleic acid primers aretargeted to homologous double-stranded, or partially double-stranded,sequences using recombinase agents, which would form D-loop structures.The invading single-stranded primers, which are part of the D-loops, areused to initiate polymerase synthesis reactions. A single primer specieswill amplify a target nucleic acid sequence through multiple rounds ofdouble-stranded invasion followed by synthesis. If two opposing primersare used, amplification of a fragment—the target sequence—can beachieved. lsRPA is described briefly in FIGS. 1 and 2.

The target sequence to be amplified, in any of the methods of theinvention, is preferably a double stranded DNA. However, the methods ofthe invention are not limited to double stranded DNA because othernucleic acid molecules, such as a single stranded DNA or RNA can beturned into double stranded DNA by one of skill in the arts using knownmethods. Suitable double stranded target DNA may be a genomic DNA or acDNA. An RPA of the invention may amplify a target nucleic acid at least10 fold, preferably at least 100 fold, more preferably at least 1,000fold, even more preferably at least 10,000 fold, and most preferably atleast 1,000,000 fold.

The target sequence is amplified with the help of recombinase agents. Arecombinase agent is an enzyme that can coat single-stranded DNA (ssDNA)to form filaments, which can then scan double-stranded DNA (dsDNA) forregions of sequence homology. When homologous sequences are located, thenucleoprotein filament (comprising the recombinase agent) strand invadesthe dsDNA creating a short hybrid and a displaced strand bubble known asa D-loop. Suitable recombinase agents include the E. coli RecA proteinor any homologous protein or protein complex from any phyla. These RecAhomologues are generally named Rad51 after the first member of thisgroup to be identified. Other recombinase agents may be utilized inplace of RecA, for example as RecT or RecO. Recombinase agents generallyrequire the presence of ATP, ATPγS, or other nucleoside triphosphatesand their analogs. It is preferred that recombinase agents are used in areaction environment in which regeneration of targeting sites can occurshortly following a round of D-loop stimulated synthesis. This willavoid a stalling of amplification or inefficient linear amplification ofssDNA caused by oscillating single-sided synthesis from one end to theother.

Naturally, any derivatives and functional analogs of the recombinaseagent above may also function itself as a recombinase agent and thesederivatives and analogs are also contemplated as embodiments of theinvention. For example, a small peptide from recA which has been shownto retain some aspects of the recombination properties of recA may beused. This peptide comprise residues 193 to 212 of E. coli. recA and canmediate pairing of ssoligos (Oleg N. Voloshin, Lijang Wang, R. DanielCamerini-Otero, Homologous DNA pairing Promoted by a 20-amino AcidPeptide Derived from RecA. Science Vol. 272 10 May 1996).

Since the use of ATPγS results in the formation of stableRecombinase-agent/dsDNA complexes that are likely incompatible withefficient amplification, it is preferable to use ATP and auxiliaryenzymes to load and maintain the Recombinase-agent/ssDNA primercomplexes. Alternatively, the limitations of the use of ATPγS may beovercome by the use of additional reaction components capable ofstripping recA bound to ATPγS from exchange complexes. This role mightbe played by helicases such as the RuvA/RuvB complex.

The terms ‘nucleic acid polymer’ or ‘nucleic acids’ as used in thisdescription can be interpreted broadly and include DNA and RNA as wellas other hybridizing nucleic-acid-like molecules such as those withsubstituted backbones e.g. peptide nucleic acids (PNAs), morpholinobackboned nucleic acids, or with modified bases and sugars.

In a preferred embodiment, RPA is performed with several auxiliaryenzymes that can promote efficient disassembly ofRecombinase-agent/dsDNA complexes after DNA synthesis initiation. Theseauxiliary enzymes include those that are capable of stimulating 3′ to 5′disassembly and those capable of supporting 5′ to 3′ disassembly.

Auxiliary enzymes include several polymerases that can displace RecA inthe 3′ to 5′ direction and can stimulate 3′ to 5′ disassembly ofRecombinase-agent/dsDNA complexes (Pham et al., 2001). These DNApolymerase include E. coli PolV and homologous polymerase of otherspecies. Normally in the life of E. coli, displacement of RecA in the 3′to 5′ direction occurs as part of SOS-lesion-targeted synthesis inconcert with SSB, sliding clamps and DNA polymerase. The polymeraseessential for this activity in E. coli is PolV, a member of the recentlydiscovered superfamily of polymerases including UmuC, DinB, Rad30 andRevl, whose function in vivo is to copy DNA lesion templates. Criticalto RPA, the in vitro 3′ to 5′ disassembly of RecA filaments cannot becatalyzed by PolI, PolIII or PolIV alone. Only PolV, in concert withSSB, has measurable ATP-independent 3′ to 5′ RecA/dsDNA disassemblyactivity. In effect, PolV pushes and removes RecA from DNA in a 3′ to 5′direction ahead of the polymerase (Pham et al., 2001; Tang et al.,2000). Inclusion of PolV or a functional homologue may improve theamplification efficiency.

Other auxiliary enzymes include a class of enzymes called helicaseswhich can be used to promote the disassembly of RecA from dsDNA. Thesepromote disassembly in both the 5′ to 3′ and 3′ to 5′ directions.Helicases are essential components of the recombination process in vivoand function to move the branch points of recombination intermediatesfrom one place to another, to separate strands, and to disassemble andrecycle components bound to DNA. After the first round ofinvasion/synthesis has occurred in RPA, two new DNA duplexes are“marked” by the presence of RecA bound over the site to which primersmust bind for additional rounds of synthesis. In such a situation dsDNAtends to occupy the high affinity site in RecA until it is activelydisplaced, either by ATP-dependent dissociation in the 5′ to 3′direction, which may be limiting, or by 3′ to 5′ dissociation by someother active process. An ideal helicase complex for stimulatingdisassembly of RecA from intermediates consists of the E. coli proteinsRuvA and RuvB. The RuvAB complex promotes branch migration, anddissociates the RecA protein, allowing RecA to be recycled (Adams etal., 1994). Normally, the RuvAB complex is targeted to recombinationintermediates, particularly Holliday junction-like structures. As itworks the RuvAB complex encircles DNA and forces RecA from the DNA in anATP-driven translocation (Cromie and Leach, 2000; Eggleston and West,2000). This RecA dissociation activity has been demonstrated usingsupercoiled dsDNA bound with RecA, which does not even possess Hollidayjunctions (Adams et al., PNAS 1994). The RuvAB complex can recognizebranched structures within the RecA coated DNA. Incorporation of RuvABinto the RPA mixture will promote the dissociation of RecA from dsDNAfollowing strand exchange and displacement, allowing renewed synthesisof the duplicated template from the same site. Additionally, the RuvABcomplex can act in concert with RuvC, which finally cuts and resolvesHolliday junctions. With RuvC added to the RPA reaction mixture,complicated structures such as Holliday junctions formed at invasionsites, can be resolved. Resolvase activity, such as that provided byRuvC, is particularly important when the targeting oligonucleotides arepartially double-stranded. In such situations reverse branch migrationcan generate Holliday junctions, which can then be resolved by theRuvABC complex, to generate clean separated amplification products.

Still other auxiliary enzymes include the E. coli RecG protein. RecG canstimulate disassembly of branch structures. In vivo this proteinfunctions to reverse replication forks at sites of DNA damage byunwinding both leading and lagging strands driving the replication forkback to generate a 4-way junction (Cox et al., 2000; Dillingham andKowalczykowski, 2001; Singleton et al., 2001). In vivo such junctionsfunction as substrates for strand switching to allow lesion bypass. Invitro RecG will bind to D-loops, and will lead to a decrease in D-loopstructures by driving reverse branch migration. RecG prefers a junctionwith double-stranded elements on either side, hence partlydouble-stranded targeting oligonucleotides, homologous to the targetingsite in both single-stranded and double-stranded regions, would beideal. This would stimulate reverse branch migration and formation of aHolliday junction, which can be resolved by the RuvABC complex. In vivoRecG and RuvAB may compete to give different outcomes of recombinationsince branch migration will be driven in both directions (McGlynn andLloyd, 1999; McGlynn et al., 2000). In both cases the proteins targetjunction DNA coated with RecA, and disassemble it in an active manner.

Other auxiliary enzymes useful in an RPA reaction mixture are those thatallow continual generation of RecA nucleoprotein filaments in thepresence of ATP and SSB. In order to allow removal of RecA at theappropriate moments, it is preferred to use ATP rather than ATPγS in anRPA reaction. Unfortunately RecA/ssDNA filaments formed with ATPspontaneously depolymerize in the 5′ to 3′ direction, and in thepresence of SSB, as required here, repolymerization will not occur atsignificant rates. The solution to this problem is the use of the RecO,RecR and possibly RecF proteins. In the presence of SSB and ATP,RecA/ssDNA filaments dissociate (Bork et al., 2001; Webb et al., 1995;Webb et al., 1997; Webb et al., 1999). If RecA/ssDNA is incubated in thepresence of RecO and RecR proteins this dissociation does not occur.Indeed the RecR protein remains associated with the filament andstabilizes the structure indefinitely. Even if ssDNA is bound by SSB, inthe presence of RecR and RecO, filaments of RecA can reassembledisplacing SSB. Thus it is possible to obviate the use of ATPγS, ifnecessary, by using ATP in the presence of RecO and RecR to maintainRecA/ssDNA filament integrity. The RecF protein interacts with the RecOand RecR system in a seemingly opposing manner. RecF competes with RecRtending to drive filament disassembly in vitro. It is likely that allthree components in vivo function together to control the generation ofinvading structures, while limiting the extent of RecA coating of ssDNA.In another preferred embodiment, RecF is included in RPA reactions at anappropriate concentration to re-capitulate the dynamics of the in vivoprocesses. In addition, RecF may facilitate dissociation of RecA-coatedintermediates after invasion has occurred.

As described, the use of ATP rather than ATPγS, and/or the use ofdisplacing polymerases and helicases (e.g. the RuvA/RuvB complex), RecO,RecR and RecF should permit exponential amplification of double-strandedDNA by driving continual regeneration of targeting sites. This method,however, remains responsive to differences in initiation rate that mightoccur at the two opposing targeting sites. Such differences may lead toa decrease in amplification efficiency, and to the production of somesingle-stranded DNA. The PCR method largely avoids these complicationsbecause temperature cycling leads to coordinated synthesis from eitherside. In another embodiment, a situation analogous to the PCR conditionjust described may be induced by using temperature sensitive (ts)mutants of RecA that are non-functional at 42° C., but function at lowertemperatures in the range 25 to 37° C. (Alexseyev et al., 1996; Hicksonet al., 1981). In this case, synthesis from either end can besynchronized by periodic lowering to the permissive temperature and thenraising the reaction to a temperature non-permissive for the mutant RecAprotein function, but permissive for the other components. By performingRPA with tsRecA mutants in combination with cycling of reactiontemperatures, the number of molecules of DNA produced can be controlled.While this will require some mechanism to provide temperature cycling,the temperatures are well below those that would require the use ofthermophile-derived proteins. Indeed, a simple chemical-based orportable low-power temperature-cycling device may be sufficient tocontrol such reaction cycles.

RPA, as all other present-day nucleic acid amplification methods,employs polymerases to generate copies of template nucleic acidmolecules. It is a necessity of most nucleic acid polymerases thatincorporation requires a free 3′-hydroxyl moiety on the terminal sugarof a short stretch of double-stranded nucleic acid adjacent to the siteof new synthesis. This stretch of double-stranded nucleic acid istypically formed on a template by a short complementary sequence, calleda primer, which serves as an initiation site for the polymerasesynthesis reaction. In some cases a 3′ modification, such as asulfydryl, may utilized to prime the synthesis reaction. The primernucleic acid, which is base-paired with the template and extended by thepolymerase, can be RNA or DNA. In vivo during genomic DNA replication,RNA primer sequences are synthesized de novo onto template DNA byprimase enzymes. Typically, for in vitro reactions the primer issupplied as a short, often chemically synthesized, single-stranded DNA(or modified DNA or RNA), and is usually referred to as anoligonucleotide primer. The primer is often of a specific sequence,although random primers can also be used. The primer is targeted tocomplementary sequences by virtue of its specific base-pairing capacity.Formation of hybrids between the oligonucleotide primer and targetnucleic acid are typically formed by incubation of the two in solutionunder conditions of salt, pH and temperature that allow spontaneousannealing.

In the case of the PCR the oligonucleotide primer is usually in vastexcess for two main reasons. First, the high concentration will driverapid annealing. Second, as the reaction proceeds through rounds ofmelting, annealing and extension the primer is consumed and becomeslimiting. PCR targeted nucleic acids are often initially double-strandedin character, and if not, become double stranded following the firstsynthetic cycle. Such double-stranded molecules cannot anneal newoligonucleotides at temperature and solvent conditions appropriate forthe catalytic activity and stability of most prokaryotic and eukaryoticproteins. Consequently, in order to allow cycles of amplification theoriginal template and the newly synthesized strands must be firstseparated before annealing can occur once again. In practice this isachieved by thermal melting. For PCR, temperatures of at least 80° C.are required for thermal melting of most double-stranded nucleic acidmolecules of lengths greater than 100 base pairs. In most PCR protocolsa temperature of 90 to 100° C. is applied to melt the DNA. Suchtemperatures allow only rare thermostable enzymes to be used. Thesepolymerases are typically derived from thermophilic prokaryotes.

The advantage of RPA is that it allows the formation of short stretchesof double-stranded nucleic acids bearing a free 3′ —OH for extensionfrom double-stranded templates without thermal melting. This is achievedby using the RecA protein from E. coli (or a RecA relative from otherphyla). In the presence of ATP, dATP, ddATP, UTP, ATPγS, and possiblyother types of nucleoside triphosphates and their analogs, RecA willform a nucleoprotein filament around single-stranded DNA. This filamentwill then scan double-stranded DNA. When homologous sequences arelocated RecA will catalyze a strand invasion reaction and pairing of theoligonucleotide with the homologous strand of the target DNA. Theoriginal pairing strand is displaced by strand invasion leaving a bubbleof single stranded DNA in the region.

RecA protein can be obtained from commercial sources. Alternatively itcan be purified according to standard protocols e.g. (Cox et al., 1981;Kuramitsu et al., 1981). RecA homologues have been purified fromthermophilic organisms including Thermococcus kodakaraensis (Rashid etal., 2001), Thermotoga maritima (Wetmur et al., 1994), Aquifexpyrophilus (Wetmur et al., 1994), Pyrococcus furiosus (Komori et al.,2000), Thermus aquaticus (Wetmur et al., 1994), Pyrobaculum islandicum(Spies et al., 2000), and Thermus thermophilus (Kato and Kuramitsu,1993). RecA has also been purified from other prokaryotes e.g.Salmonella typhimurium (Pierre and Paoletti, 1983), Bacillus subtilis(Lovett and Roberts, 1985), Streptococcus pneumoniae (Steffen andBryant, 2000), Bacteroides fragilis (Goodman et al., 1987), Proteusmirabilis (West et al., 1983), Rhizobium meliloti (Better and Helinski,1983), Pseudomonas aeruginosa (Kurumizaka et al., 1994), from eukaryotese.g. Saccharomyces cerevisiae (Heyer and Kolodner, 1989), Ustilagomaydis (Bennett and Holloman, 2001), including vertebrates e.g. HumanRad51 (Baumann et al., 1997) and Xenopus laevis (Maeshima et al., 1996),as well as plants including broccoli (Tissier et al., 1995).

For clarity of description, leading strand Recombinase-PolymeraseAmplification method (lsRPA) can be divided into four phases.

1) Sequence targeting

RPA is initiated by targeting sequences using synthetic oligonucleotidescoated with RecA, or a functional homologue. In order to permitexponential amplification two such synthetic oligonucleotides would beemployed in a manner such that their free 3′-ends are orientated towardone another. Nucleoprotein filaments comprising these oligonucleotidesand RecA protein will identify targets in complex DNA rapidly andspecifically. Once targeted the RecA protein catalyses strand exchangesuch that D-loop structures are formed. It may be necessary to use ATPrather than ATPγS in the procedure for efficient amplification. If ATPis used, RecO, RecR, and/or RecF, molecules may prove essential forefficient amplification.

2) Initiation of DNA synthesis

DNA polymerases will detect and bind to the hybrid between the invadingoligonucleotides and the template DNA and initiate DNA synthesis fromthe free 3′-hydroxyl exposed in the hybrid. Exposure of this3′-hydroxyl, and subsequent DNA synthesis, will likely requiredisassembly of RecA protein from the double-stranded hybrid formed bystrand exchange. To attain this disassembly it will probably benecessary to employ ATP, which can support spontaneous disassembly ofRecA from invasion complexes. Additionally disassembly can bestimulated/enhanced by the use of other proteins contained within thereaction mixture such as RuvA, RuvB, RuvC, recG, other helicases, orother stimulatory components, which can act to strip RecA from thestrand exchange product.

3) Strand displacement DNA synthesis and replicon separation.

As the DNA polymerases synthesize complementary copies of template DNAsusing the free 3′-hydroxyls of invading oligonucleotides, or theirpartly extended products, the polymerases displace single-stranded DNAs,which may be coated with single strand binding proteins (SSB) includedin the reaction. In an ideal configuration, invasion of oligonucleotidesat both ends of the target nucleic acid sequence will occur in similartimeframes, such that two polymerases on the same template nucleic acidwill initially progress toward one another. When these extendingcomplexes meet one another, the original template should simply fallapart, and the polymerases will continue to synthesize without a needfor strand displacement, now copying SSB-bound ssDNA template. Becauseof steric hinderance, polymerases may become dissociated from thetemplate temporarily when the polymerases meet to permit the separationof the two template strands

4) Completion of synthesis and re-invasion.

Once the template strands have separated, polymerases can complete theextension to the end of the template (or past the sequence acting as thesecond, facing, targeting site if the initial template is longer thanthe desired product). To permit exponential amplification it isnecessary for new products to be targeted and replicated in a mannersimilar to the original templates, that is from both targeted ends. Thenewly synthesized targeted site will be freely available to targetingRecA/oligonucleotide filaments. The site initially used to primesynthesis should also have been freed as a consequence of the use ofconditions in the reaction that favor disassembly of RecA from strandexchange products. Providing the re-invasion at this latter site occursin less time than it takes the polymerase to synthesize past the secondtargeting site, be primed at that second site, and return to the firstsite, then single-stranded DNA will not be the primary product andexponential amplification will occur. Having multiple syntheticcomplexes operating on the same template raises the possibility thatvery short amplification times can be achieved.

Recombinase-Polymerase Amplification (RPA) Using Simultaneous Leadingand Lagging Strand Synthesis

In our description of (leading strand RPA) lsRPA we detail amulti-component system with the capacity to regenerate targetingsequences thus permitting exponential amplification of double-strandedDNA. Unlike the Zarling method, lsRPA avoids the linear production ofsingle-stranded DNA. There is another approach to solving this problemthat completely avoids the possibility of single-stranded products and arequirement for simultaneous end initiation. This method necessarilyinvolves a more complex reaction mixture. Nevertheless all of therequired components are now well understood and should be amenable toassembly into a single system. This system will recapitulate eventsoccurring during the normal replication cycle of cells to permit coupledleading and lagging strand synthesis. This method, leading/laggingstrand RPA is described briefly in FIGS. 1 and 3.

During normal replication in vivo, double-stranded DNA is simultaneouslyseparated into 2 strands and both are copied to give 2 new molecules ofdouble-stranded DNA by a replication machine. This ‘machine’ couplesconventional 5′ to 3′ leading strand synthesis with lagging strandsynthesis, in which short RNA primers are synthesized onto templatenucleic acids by primase enzymes. During lagging strand synthesis, shortfragments of DNA are produced, called Okazaki fragments, which areligated together to form contiguous lagging strands. This simultaneousleading-strand/lagging-strand synthesis is responsible for duplicationof the entire genome of prokaryotic and eukaryotic organisms alike. Theessential components of this system have been identified andcharacterized biochemically. The components can be assembled in vitro toachieve a more efficient amplification than possible using onlyleading-strand synthesis.

The essential components of the replication ‘machine’ are now wellcharacterized for E. coli. This machine comprises the PolIII holoenzyme(Glover and McHenry, 2001; Kelman and O'Donnell, 1995) and the primosome(Benkovic et al., 2001; Marians, 1999). The PolIII holoenzyme is made upof ten polypeptide components. Each holoenzyme contains two,asymmetrically oriented, core structures, each consisting of apolymerase (α subunit) and two additional core components the ε subunit,which possesses 3′ to 5′ exonuclease activity, and the θ subunit. Inaddition to the core complex another set of polypeptides provide theholoenzyme with processivity and couple leading and lagging strandsynthesis. The β-dimer sliding clamp encircles the template DNA affixingthe complex to the template with extremely high affinity. The slidingclamp loaded onto DNA by the DnaX clamp loader comprising the τ₂γδδ′χψpolypeptide subunits.

For clarity of description, the RPA method can be divided into fourphases. In reality all phases will occur simultaneously in a singlereaction.

1) Sequence targeting

RPA is initiated by targeting sequences using synthetic oligonucleotidescoated with RecA, or a functional homologue. Such nucleoproteinfilaments will identify targets in complex DNA rapidly and specifically.Once targeted, the RecA protein catalyses strand exchange such that aD-loop structure is formed. It may be necessary to use ATP rather thanATPγS in the procedure for efficient amplification. The linkage ofleading and lagging strand syntheses however may obviate the requirementfor very rapid RecA stripping after initiation of synthesis. If ATP isused, RecO, RecR, and RecF molecules may prove essential for efficientamplification.

2) Primosome assembly

Primosomes can be assembled at D-loops. Normally, D-loop structures areformed by RecA as part of the mechanism to rescue damaged DNA in vivo,or during other forms of recombination. The purpose of the combinedaction of RecA-mediated strand exchange and primosome assembly is togenerate a replication fork. A replication fork is the nucleoproteinstructure comprising the separated template DNA strands and thereplisome. The replisome consists of the polymerase holoenzyme complex,the primosome, and other components needed to simultaneously replicateboth strands of template DNA. Primosomes provide both the DNA unwindingand the Okazaki fragment priming functions required for replication forkprogression.

Primosome assembly has been studied intensively through genetic andbiochemical analysis in E. coli. The minimal set of polypeptidesrequired for this process is well known and exist as purifiedcomponents. The primosome assembly proteins are PriA, PriB, PriC, DnaT,DnaC, DnaB and DnaG. These proteins have been shown sufficient toassemble a primosome complex on bacteriophage DX174 DNA in vitro(Kornberg and Baker, 1992; Marians, 1992). PriA binds to the primosomeassembly site (PAS) on the ΦX174 chromosome. Then PriB, DnaT, and PriCbind sequentially to the PriA-DNA complex. PriB appears to stabilizePriA at the PAS and facilitate the binding of DnaT (Liu et al., 1996).PriC is only partially required for the full assembly reaction. Omissionof PriC from the reaction will lower priming 3 to 4 fold (Ng andMarians, 1996a; Ng and Marians, 1996b). The function of PriC in thebacterium is genetically redundant to PriB. DnaC then loads DnaB intothe complex in an ATP-dependent fashion. This PriABC-DnaBT complex iscompetent to translocate along the chromosome. The DnaG primase caninteract transiently with the complex to synthesize RNA primers.

During replication in E. coli, DnaB and DnaG function as a helicase andprimase respectively. These two components are continually required inassociation with the PolIII holoenzyme to synthesize primers for theOkazaki fragments. Hence, DnaB and DnaG are the core components of themobile primosome associated with the replication fork. The otherprimosome components described are essential for assembly of theprimosome onto DNA, and for associating a dimeric polymerase. Theprimosome assembly proteins are required for the re-establishment ofreplication forks at recombination intermediates formed by RecA andstrand exchange. PriA can initiate assembly of a replisome, competentfor DNA synthesis, on recombination intermediates. It is possible totarget D-loops in vitro with a mixture of PriA, PriB and DnaT, which arethen competent to incorporate DnaB and DnaC. Once a primosome has beenformed at the D-loop, all that remains to initiate replication is toload a holoenzyme complex to the site.

3) Fork assembly and initiation of DNA synthesis

Replication forks will assemble at the site of primosome assembly. Thepresence of a free 3′-end on the invading strand of the D-loopstimulates the DnaX clamp loader complex detailed earlier to assemble aβ-dimer at this site to act as a sliding clamp. The holoenzyme and τcore units are joined together by the scaffold τ subunit. The τ subunitalso has interaction surfaces for the β-dimer, for the clamp loader, andfor the DnaB helicase component of the primosome. These multipleinteractions are necessary to coordinate synthesis of both leading andlagging strands using the 2 asymmetrically joined core polymerasecomplexes.

The primosomal primase, DnaG, synthesizes a short RNA primer onto theunwound lagging strand DNA template. In the presence of the holoenzyme,the clamp loader recognizes the RNA/DNA duplex and loads a secondβ-dimer clamp onto this site. The presence of an active primosome andthe interaction of the τ subunit with DnaB are critical to ensuresimultaneous leading/lagging strand synthesis. Without this interactionthe polymerase will move away from the primosome site without coupling.

A replication fork is now assembled. Synthesis of both leading andlagging strand will now occur simultaneously, and the DnaB helicase willseparate template strands ahead of the oncoming holoenzyme. The laggingstrand holoenzyme core will generate Okazaki fragments of 1 to 2kilobases in length. Once the lagging strand polymerase encounters theprevious RNA primer, it dissociates from the β-clamp and synthesis isinitiated from a newly assembled clamp loaded in the vicinity of thefront of the leading strand. The same lagging strand holoenzyme corewill be re-used since it is physically tethered to leading strand core.

There is a dynamic interaction between β-dimer clamps, core subunits andclamp loaders. Their affinities can switch depending upon the physicalcircumstances. The β-dimer that has been ‘abandoned’ at the end of theOkazaki fragments may be recycled via active removal by clamp loaders,or excess δ subunit that may be present.

The RNA primers at the ends of Okazaki fragments are removed by the 5′to 3′ exonuclease activity of DNA polymerase I. DNA ligase then joinsthe Okazaki fragments together forming a continuous lagging strand.

4) Fork meeting and termination

In RPA, replication is initiated at two distant sites and thereplication forks are oriented toward each other. As replication forksconverge the two original template strands will dissociate from oneanother as they become separated entirely both behind, and in front, ofeach fork. The leading strand core of each fork will then completesynthesis, the remaining RNA primers will be processed, and the finalproducts will be two double-stranded molecules. We can reasonably expectto amplify DNA's on the order of several Megabases (Mb) by such anapproach. In this disclosure, megabase also encompasses megabasepairs.Based on the known synthetic rate of the PolIII holoenzyme we can expectthe replication forks to proceed at a rate of approximately 1 Mb/1000seconds, i.e., approximately 15 to 20 minutes per cycle for a 1 Mbfragment.

The final consideration is the mechanism by which rapid exponentialamplification of DNA will be achieved. The key to this process will beto allow efficient reinvasion of the targeting sites by the use ofmixtures of helicases, resolvases and the RecO, RecR, and RecF proteins.Under appropriate conditions reinvasion and primosome assembly should bepossible shortly after a holoenzyme has moved away from thefork-assembly site. Continual invasions should present no problems sincethe DNA will simply become branched at many points. Each branch willnaturally resolve as it encounters the oncoming fork. Under theseconditions it may be possible to achieve enormous amplification in timessimilar to the time taken to replicate the DNA only once. It may becritical however to limit the concentrations of targetingoligonucleotides to avoid nucleotide depletion prior to the completionof synthesis.

In addition to the holoenzyme complex, the replication machine employsanother complex known as the primosome, which synthesizes the laggingstrand and moves the replication fork forwards. The primosome complexcomprises a helicase encoded by DnaB and a primase encoded by DnaG.Finally, in addition to the proteins of the holoenzyme and primosome,replication requires the activity of single-stranded DNA binding protein(SSB), E. coli DNA polymerase I and DNA ligase. These latter twocomponents are required to process Okazaki fragments.

Nested RPA:

In another embodiment, RPA amplification may be performed in a processreferred to herein as “nested RPA.”

A difficulty in detecting a rare sequence is that there can be a highratio of non-target to target sequence. The ability of a RPA todiscriminate between target and non-target DNA and amplify only targetsequences is a key aspect of improved sensitivity. Discriminationbetween non-target and target is a reflection of the specificity of theprimers and reaction conditions. The more specific a reaction is thegreater the relative amount of the specific target sequence that isproduced and the easier that product is to detect. An increase inspecificity can, therefore, increase sensitivity as well.

The need for improved sensitivity and specificity can be addressed byusing nested RPA. The nested RPA involves a first RPA of a first regionof DNA. Then the reaction mixture is diluted, for example, by 10, 20,30, 40, 50, 75, or 100 fold or more to reduce the concentration of thefirst primer pair, and a second primer pair is introduced into thereaction mixture and RPA repeated. According to one embodiment of theinvention, the second primer pair is designed to be internal to thefirst primer pair to amplify a subsequence of the first RPA product. Themethod increases specific amplification, i.e., reduces non-specificbackground amplification products and therefore increases sensitivity.Such non-specific amplification products, although they arise by virtueof fortuitous partial homology to the flanking primers, are unlikely toalso have sufficient homology to the nested primers to continue toamplify. Detection and specificity of RPA may be further improved bylabeling one or both of the second primer pair such that only primersamplified with one or both of the second primer pair is detected.

Nested RPA is not limited to the use of two sets of primer. Naturally,more sets of primers may be used to increase specificity or sensitivity.Thus, three, four or five pairs of primers may be used. Furthermore, thedifferent sets of primers, as another embodiment of the invention, mayshare common primers as illustrated in FIG. 4.

In FIG. 4, the primer sets are designed to be used sequentially. Forexample, a first RPA is performed with primer set 1, a second RPA usingthe amplified product of the first RPA is performed with a primer set 2,a third RPA using the amplified product of the second RPA is performedwith a primer set 3, a fourth RPA using the amplified sequence of thethird RPA is performed with a primer set 4, and finally, a fifth RPA isperformed using the amplified product of the fourth RPA is performedwith a primer set 5. In this case, primer set 1, 2, and 3, share acommon primer-primer (a). Primer 3, 4, and 5 share a commonprimer-primer (b).

Nested RPA may be performed using any of the two RPA methods describedas well as a combination of the two methods in any particular order.That is, RPA may be performed solely by leading strand RPA, solely byleading and lagging strand RPA, or a combination of leading strand RPAand leading and lagging strand RPA in any particular order.

One benefit of any of the RPA methods of the invention is the size ofthe amplified product. While current methods of amplification such asPCR are limited to an upper limit of about 10 Kb, RPA methods arecapable of amplifying regions of nucleic acids of up to hundreds ofmegabases. For leading/lagging strand RPA, the sizes of a targetsequence to be amplified can be hundreds of megabases, such as, forexample, less than 500 megabases, less than 300 megabase, less than 100megabase, less than 70 megabase, less than 50 megabase, less than 25megabase, less than 10 megabase, less than 5 megabase, less than 2megabass, less than one megabase, less than 500 kb, less than 200 kb,less than 100 kb, less than 50 kb, less than 25 kb, or less than 10 kb,less than 5 kb, less than 2 kb, less than 1 kb. For lsRPA, the sizes ofa target sequence can be in the megabase range such as, less than 5megabase, less than 2 megabass, less than one megabase, less than 500kb, less than 200 kb, less than 100 kb, less than 50 kb, less than 25kb, or less than 10 kb, less than 5 kb, less than 2 kb, less than 1 kb.

Selection of RPA Reagents and Reaction Parameters

The details of leading strand RPA, leading and lagging strand RPA, andnested RPA were listed above. This section will describe the selectionof reagents and parameter for any of the three methods discussed above.

One benefit of RPA is that the amplified product of RPA is doublestranded DNA which could be used for other molecular biology procedures.Thus, RPA may be combined with other methods in molecular biology. Forexample, the starting material for RPA may be a PCR amplified fragment.Alternatively, the product of an RPA may be used for PCR.

If necessary, the RPA products in any of the methods of the inventionmay be purified. For example, in the nested RPA method, the amplifiedproduct may be purified after each RPA step before a subsequent RPAstep. Methods of purification of nucleic acids are known in the art andwould include, at least, phenol extraction, nucleic acid precipitation(e.g., with salt and ethanol), column chromatography (e.g., sizeexclusion, ionic column, affinity column and the like) or anycombination of these techniques.

As discussed, the primers used in RPA may be “double stranded” or“capable of forming double stranded structures.” These terms refer toDNA molecules that exist in a double stranded condition in a reactionsolution such as a RPA reaction solution or a PCR reaction solution. Thecomposition of a PCR solution is known. The composition of a RPAreaction is listed in this detailed description section and in theExamples.

The primers may have a single stranded region for hybridization to thetarget DNA in the presence of a recombinase agent. The single strandedregion may be, for example, about 10 bases about 15 bases, about 20bases, about 25 bases, about 30 bases, about 40 bases, and about 50bases. Even longer regions such as about 75 bases, about 100 bases,about 150 base or more may be used but it is not necessary. The choiceof single stranded regions will depend on the complexity of the startingnucleic acid so that for example, a human genome may require a longerprimer while a plasmid may require a much shorter primer.

The two strands of nucleic acid in a double stranded DNA need not becompletely complementary. For example, the double-stranded region of adouble-stranded DNA may differ by up to 1% in sequence. That is, thesequence of two strands of nucleic acid may differ by one base in onehundred bases and would still exist in a double stranded condition insolution. Nucleic acids with 1% difference in their complementarysequence are contemplated as double stranded DNA for the purposes ofthis disclosure.

In addition, the target nucleic acid (i.e., the nucleic acid to beamplified by the RPA methods of the invention) may be partially doublestranded and partially single stranded. For example, nucleic acid in anyof the configuration of FIG. 5 would be suitable as a target nucleicacid of the invention. As discussed, the target nucleic acid may be RNA.RNA can be converted to double-stranded cDNA using known methods and thedouble-stranded cDNA may be used as the target nucleic acid. As shown ifFIG. 5, the template nucleic acid may have any combination of endsselected from 3′ overhang, 5′ overhang, or blunt ends.

The lsRPA method of the invention comprises at least the followingsteps. First, a recombinase agent is contacted to two nucleic acidprimers (referred to herein as a first and a second primer) to form twonucleoprotein primers (referred to herein as a first nucleoproteinprimer and a second nucleoprotein primer).

Second, the first and second nucleoprotein primers are contacted to thetemplate nucleic acid to form a double stranded structure at a firstportion of the first strand and a second double stranded structure at asecond portion of the second strand. The two primers are designed sothat when hybridized, they are pointed at each other as illustrated inFIG. 6A. Alternatively, primer 1 and primer 2 may hybridize differenttarget nucleic acids as illustrated in FIG. 6B.

Third, the nucleoprotein primers are extended at their 3′ ends togenerate a first and a second double stranded nucleic acid (FIG. 7A).Where the primers are hybridized to different target nucleic acids, theelongation of the primers will generate displaced strands (FIG. 7B). Inthis case, the two displaced strands that result from primer elongationmay hybridize and form a new double stranded template nucleic acid (FIG.7C).

Step two and three are repeated until the desired degree ofamplification is reached. The process is a dynamic process in thatprimer hybridization to the target nucleic acid and elongation areallowed to proceed continuously. One advantage of this invention is thatthe amplification is performed continuously without the need fortemperature cycling or enzyme addition after initiation of the reaction.

In an embodiment, steps two and three are repeated at least 5 times.Preferably, it is repeated at least 10 times. More preferably, it isrepeated at least 20 times, such as at least 30 times. Most preferably,the two steps are repeated at least 50 times. For multiple repetitionsof the amplification step (e.g., step 2 and 3) a RPA of the invention ispreferably started with a primer to target nucleic acid ration of atleast 100 to 1, preferably at least 300 to 1, and most preferably atleast 1000 to 1. That is, there are at least 100, 300 or 1000 copies ofthe primer per copy of a target nucleic acid.

In an optional step, after a sufficient round of amplification,additional components may be added to the reaction after a period oftime to enhance the overall amplification efficiency. In one embodiment,the additional components may be one or more of the following:recombinase agents, one or more primers, polymerase, and one or more ofthe additional agents (discussed in a separate section below).

In a preferred embodiment, a small fraction of a first RPA reaction isused as a supply of template DNA for subsequent rounds or RPAamplification. In this method, a first RPA amplification reaction isperformed on a target nucleic acid. After the first RPA reaction, asmall fraction of the total reaction is used as a substitute of thetarget nucleic acid for a subsequent round of RPA reaction. The fractionmay be, for example, less than about 10% of the first reaction.Preferably, the fraction may be less than about 5% of the firstreaction. More preferably, the fraction may be less than 2% of the firstreaction. Most preferably, the fraction may be less than 1% of theinitial reaction.

The primer used in RPA is preferably DNA although PNA, and RNA are alsosuitable for use as primers. It is noted that in fact, in DNAreplication, DNA polymerases elongate genomic DNA from RNA primers.

Synthetic oligonucleotides may serve as DNA primer and can be used assubstrates for formation of nucleoprotein filaments with RecA or itshomologues. Sequences as short as 15 nucleotides are capable oftargeting double-stranded DNA (Hsieh et al., 1992). Sucholigonucleotides can be synthesized according to standardphosphoroamidate chemistry, or otherwise. Modified bases and/or linkerbackbone chemistries may be desirable and functional in some cases.Additionally oligonucleotides may be modified at their ends, either 5′or 3′, with groups that serve various purposes e.g. fluorescent groups,quenchers, protecting (blocking) groups (reversible or not), magnetictags, proteins etc. In some cases single-stranded oligonucleotides maybe used for strand invasion, in others only partly single strandednucleic acids may be used, the 5′ stretch of sequence of an invadingnucleic acid being already hybridized to an oligonucleotide.

In another embodiment of the invention, the primers may comprise a 5′region that is not homologous to the target nucleic acid. It should benoted that the processes of the invention should be functional even ifthe primers are not completely complementary to the target nucleic acid.The primers may be noncomplementary by having additional sequences attheir 5′ end. These additional sequences may be, for example, thesequence for a restriction endonuclease recognition site or the sequencethat is complementary to a sequencing primer. The restrictionendonuclease recognition site may be useful for subsequent cleavage ofthe amplified sequence. The use of restriction endonuclease that cleavesnucleic acid outside the restriction endonuclease recognition site isalso contemplated. The sequence that is complementary for a sequencingprimer may allow rapid DNA sequencing of the amplified product usingcommercially available primers or commercially available sequencingapparatus.

Formation of nucleoprotein filaments can be performed by incubation ofthe primer (oligonucleotides) with RecA protein or its homologues in thepresence of ATP, and auxiliary proteins such as RecO, RecR and RecF.When incubated at 37° C. in RecA buffer (20 mM Tris-HCl pH 7.5, 10 mMMgCl₂, 2 mM ATP, 2 mM DTT and 100 ug/ml Bovine Serum Albumin), RecA willform helical filaments on ssDNA with 6 protomers per turn. The DNA islocated within the interior of the protein helix. In the presence ofdsDNA, the RecA/ssDNA nucleoprotein filament can scan DNA at rates of atleast 10⁷ bp per hour. The mode of scanning is unclear but it is at aspeed (>10³ bp per second) that it may involve only the initial few basepairs that can be easily accessed along one face of the major groove.Successful binding may result in a transition to a triple-helicalintermediate, which is then followed by strand invasion and displacementto form a D-loop. Such joint molecules can be formed under similarcondition to those described above for formation of helical filaments,and hence in the presence of ssDNA, the homologous dsDNA, RecA, ATP,auxiliary proteins and suitable buffer and temperature conditions, jointmolecules will form spontaneously. If ATP is used the assembly isreversible and will reach equilibrium, but RecA/ssDNA filaments can bestabilized, even in the presence of SSB, by the auxiliary proteins RecOand RecR. In the case of thermostable proteins the temperature ofincubation can be higher. If a renewable supply of ATP is required astandard ATP regeneration system can be included in the reaction.

DNA polymerases can use the free 3′-hydroxyl of the invading strand tocatalyze DNA synthesis by incorporation of new nucleotides. A number ofpolymerases can use the 3′-hydroxyl of the invading strand to catalyzesynthesis and simultaneously displace the other strand as synthesisoccurs. For example E. coli polymerase II or III can be used to extendinvaded D-loops (Morel et al., 1997). In addition, E. coli polymerase Vnormally used in SOS-lesion-targeted mutations in E. coli can be used(Pham et al., 2001). All of these polymerases can be rendered highlyprocessive through their interactions and co-operation with the β-dimerclamp, as well as single stranded DNA binding protein (SSB) and othercomponents. Other polymerases from prokaryotes, viruses, and eukaryotescan also be used to extend the invading strand.

In another embodiment of the invention, the primer may be partiallydouble stranded, partially single stranded and with at least one singlestranded 3′ overhang. In this embodiment, the primer may comprise ainvading strand and a non-invading strand as shown in FIG. 8A. In thiscase, after the invading strand is hybridized to the target DNA andelongated, it serves as a target nucleic acid for a second primer asshown in FIG. 8B. The elongation of the second primer would displace thenoninvading strand as shown in FIG. 8C. In this embodiment, as thetarget nucleic acid is amplified, the non-invading strand of primer 1 isdisplaced. If both primer one and primer two are partly double strandedprimers, then the non-invading strands of both primer one and primer twowill accumulate in solution as the target nucleic acid is amplified.

In one embodiment of the invention, at least two of the primers in a RPAreaction are partially double stranded and partially single strandedeach generated by the hybridization of an invading strand and anon-invading oligonucleotide strand, which possess sequences ofsufficiently complementary that they form a double stranded region.Preferably, the two oligonucleotide strands are sufficientlycomplementary over the releavant region that they can form a doublestranded structure in RPA reaction conditions.

In an embodiment of the invention, the primers, includingsingle-stranded and partially double-stranded primers, are labeled witha detectable label. It should be noted that a fluorescence quencher isalso considered a detectable label. For example, the fluorescencequencher may be contacted to a fluorescent dye and the amount ofquenching is detected. The detectable label should be such that it doesnot interfere with an elongation reaction. Where the primer is partiallydouble stranded with an invading strand and a non-invading strand, thedetectable label should be attached in such a way so it would notinterfere with the elongation reaction of the invading strand. Thenon-invading strand of a partially double stranded primer is notelongated so there are no limitations on the labeling of thenon-invading strand with the sole exception being that the label on thenon-invading strand should not interfere with the elongation reaction ofthe invading strand. Labeled primers offer the advantage of a more rapiddetection of amplified product. In addition, the detection ofunincorporated label, that is, labeled oligonucleotides that have notbeen extended, will allow the monitoring of the status of the reaction.

Monitoring a RPA reaction may involve, for example, removing a fractionof an RPA reaction, isolating the unincorporated fraction, and detectingthe unincorporated primer. Since the size of an unincorporated primermay be less than 50 bp, less than 40 bp, less than 30 bp or less than 25bp, and the size of the amplified product may be greater than 1 Kb,greater than 2 Kb, greater than 5 Kb, or greater than 10 Kb, there is agreat size difference between the incorporated and unincorporatedprimer. The isolation of the unincorporated primer may be performedrapidly using size exclusion chromatography such as, for example, a spincolumn. If a primer is labeled, a monitor procedure comprising a spincolumn and a measurement (e.g., fluorescence or radioactivity) can beperformed in less than one minute. Another alternative for separatingelongated primers from unelongated primers involve the use of PAGE. Forexample, the elongated primer may be separated from the unelongatedprimer by gel electrophoresis in less than 5 minutes. Yet anotheralternative for separating elongated primers involves the use ofimmobilized oligonucleotides. For example oligonucleotides homologous tosequences found uniquely within the amplified DNA sequence can be usedto capture nucleic acids produced by primer elongation specifically.These capturing oligonucleotides immobilized on a chip, or othersubstrate. Capture of the elongated oligonucleotides by the capturingoligonucleotides can be performed by RecA protein mediated methods, orby traditional solution hybridizations if necessary.

In another embodiment of the invention, a double stranded primer may belabeled such that the separation of the two strands of the primer may bedetected. As discussed above, after multiple rounds of elongation, theinvading strand and the noninvading strands of a partially doublestranded primer is separated. After this separation, the non-invadingstrand does not participate in the RPA reaction. This characteristic maybe used to detect and monitor a RPA reaction in a number of ways.

In this application, the detectable label may be a fluorescent label oran enzyme and the label quencher (also referred to as the labelinhibitor) may be a fluorescence quencher or an enzyme inhibitor. Inthese cases, the label is detected by fluorescence or enzyme inhibition.The delectability of the label would be the fluorescence if afluorescent label is used or enzyme activity if an enzyme is used.

In the first method, the invading strand may be labeled with a label andthe non-invading strand may be labeled with a detectable label quencher.The label, in the proximity of the label quencher (label inhibitor) onthe partially double stranded primer would not be highly detectable.After RPA, the invading strand would be separated from the noninvadingstrand and thus, the label and the label quencher would be separated.The separation would cause the label to be more detectable. Thus, RPAreactions may be monitored by measuring the increases in the amount ofdetectable label.

The second method is similar to the first method except that theinvading strand is modified with a label quencher while the noninvadingstrand is modified with a label. Then RPA is allowed to proceed with theresult (same as method 1) of the label being separated from the labelquencher. Thus, the overall delectability of the label would increase.

The third method involves labeling the noninvading strand of one doublestranded primer with a label. In addition, the noninvading strand of asecond double stranded primer is labeled with a label quencher. The twonon-invading stands are designed to be complementary to each other. Inthis configuration, the RPA reaction is initially fluorescent. As theRPA reaction progresses, the two noninvading strands are displaced intosolution and they hybridize to each other because they are designed tobe complementary. As they hybridize, the label and the label quencherare brought into proximity to each other and the fluorescence of thereaction is decreased. The progress of the RPA reaction may be measuredby monitoring the decrease in label detectability.

In a fourth method, the noninvading strands of a first and second doublestranded primers are labeled with a first label and a second label. Thetwo noninvading strands are also designed to be complementary to eachother. As in the third method, after RPA, the two noninvading strandsare hybridized to each other and the proximity of the two labels will bea reflection of the progress of the RPA reaction. The proximity of thetwo labels may be determined, for example, by direct observation or byisolation of the non-invading strands. As discussed above, isolation ofprimers and other small nucleic acids can be accomplished by sizeexclusion columns (including spin columns) or by gel electrophoresis.

In another embodiment of the invention, the non-invading strand of oneor both of the primers is homologous to a second region of nucleic acidsuch that the primer can hybridize to and primer DNA synthesis at thesecond region of nucleic acid. Using this method, a second RPA reactionusing the noninvading stand from the primer of a first RPA may bestarted. The product of the second RPA may be monitored to determine theprogress of the first RPA.

In yet another embodiment of the invention, the non-invading strand isdetected by a biosensor specific for the sequence of the non-invadingstrand. For example, the biosensor may be a surface with a nucleic acidsequence complementary to the non-invading strand. The biosensor maymonitor a characteristic that results from the binding of thenon-invading strand. The characteristic may be a detectable label.

Suitable detectable labels for any of the methods of the inventioninclude enzymes, enzyme substrates, coenzymes, enzyme inhibitors,fluorescent markers, chromophores, luminescent markers, radioisotopes(including radionucleotides) and one member of a binding pair. Morespecific examples include fluorescein, phycobiliprotein, tetraethylrhodamine, and beta-gal. Bind pairs may include biotin/avidin,biotin/strepavidin, antigen/antibody, ligand/receptor, and analogs andmutants of the binding pairs.

The recombinase agent of the invention may be RecA, RadA, RadB, Rad 51or a functional analog or homologues of these proteins. If desired, therecombinase may be a temperature-sensitive (referred to herein as “ts”)recombinase agent. If a ts recombinase is used, the RPA reaction may bestarted at one temperature (the permissive temperature) and terminatedat another temperature (the non permissive temperature). Combinations ofpermissive temperatures may be, for example 25° C./30° C., 30° C./37°C., 37° C./42° C. and the like. In a preferred embodiment, the tsprotein is reversible. A reversible ts protein's activity is restoredwhen it is shifted from the nonpermissive temperature to the permissivetemperature.

In a preferred embodiment, the RPA is performed in the presences of ATP,an ATP analog, or another nucleoside triphosphate. The ATP analog maybe, for example, ATPγS, dATP, ddATP, or another nucleoside triphosphateanalog such as UTP.

Other useful reagents that may be added to an RPA reaction includenucleotide triphosphates (i.e., dNTPs such as dATP, dTTP, dCTP, dGTP andderivatives and analogs thereof) and a DNA polymerase. Other usefulreagents useful for leading/lagging RPA include NTPs (ATP, GTP, CTP, UTPand derivatives and analogs thereof). One advantage of the RPA reactionis that there is no limit on the type of polymerase used. For example,both eukaryotic and prokaryotic polymerases can be used. Prokaryoticpolymerase include, at least, E. coli pol I, E. coli pol II, E. coli polIII, E. coli pol IV and E. coli poly. Eukaryotic polymerase include, forexample, multiprotein polymerase complexes selected from the groupconsisting of pol-α, pol-β, pol-δ, and pol-ε.

In another embodiment of the invention, the RPA process is performed inthe presence of an accessory component to improve polymeraseprocessivity or fidelity. Both eukaryotic and prokaryotic accessorycomponents may be used. Preferably, the accessory component is anaccessory protein is from E. coli. Useful accessory proteins includesingle-strand binding protein, helicase, topoisomerase and resolvase.Other useful accessory proteins include a sliding clamp selected fromthe group consisting of an E. coli β-dimer sliding clamp, an eukaryoticPCNA sliding clamp and a T4 sliding clamp gp45. Other accessorycomponents include a DNA Polymerase III holoenzyme complex consisting ofβ-Clamp, DnaX Clamp Loader, and the Polymerase Core Complex. Still otheraccessory component include RuvA, RuvB, RuvC and RecG. The propertiesendowed by the use of additional components will likely enable theamplification of large DNAs not previously successfully targeted bycurrent methods such as PCR.

In another embodiment, the RPA is performed in the presence of agentsused to stabilize a RecA/ssDNA nucleoprotein filaments. For example, theagent may be RecR, RecO, RecF or a combination of these proteins. Otheruseful agents include PriA, PriB, DnaT, DnaB, DnaC, and DnaG.

One benefit of the present invention is that the RPA reaction may beperformed at reduced temperatures compared to a PCR reaction. Forexample, the RPA process may be performed between 20° C. and 50° C.Preferably, the RPA process is performed at less than 45° C. Morepreferably, the RPA process may be performed at less than 40° C. Evenmore preferably, the RPA process may be performed at less than 35° C.Most preferably, the RPA process may be performed at less than 30° C.One of the reasons that the RPA process can be performed at thesereduced temperatures is because RPA may be performed without temperatureinduced melting of the template nucleic acid. Further, unlike PCR,absolute temperature control is not required and the temperature canfluctuate without adversely affecting RPA. For example, the amount offluctuation may be anywhere within the temperatures specified above. Thetemperature necessary for melting of double stranded DNA also contributeto premature enzyme inactivation, a disadvantage absent in the methodsof this invention.

RPA may be performed to test for the presences or absences of agenotype. The genotype tested may be associated with a disease or apredisposition to a disease. Alternatively, the genotype may beassociated with a normal phenotype or a phenotype that confers specialresistance to a disease. The genotype as disclosed above may be anystandard genetic variant such as a point mutation, a deletion, aninsertion, an inversion, a frameshift mutation, a crossover event, orthe presence or absences of multiple copies of a genetic sequence (e.g.,the presences of minichromosomes).

One method of detecting a genotype is to detect the distance between aprimer pair in an RPA reaction. The distance between a primer pair isreflected by the size of the amplified sequence. In that method, the twoprimers are selected such that it spans a target region such as, forexample, a gene. Then RPA is performed using the primer pair and the RPAproduct is analyzed. The analysis may involve to determining the size orsequence of the amplified product. Methods of determining the size of aDNA sequence, including at least techniques such as agarose gels, PAGEgels, mass spectroscopy, pulsed field gels, gene chips, sucrosesedimentation and the like are known. There are many DNA sequencingmethods and their variants, such as the Sanger sequencing using dideoxytermination and denaturing gel electrophoresis (Sanger, F., Nichlen, S.& Coulson, A. R. Proc. Natl. Acad. Sci. U.S.A. 75, 5463-5467 (1977)),Maxam-Gilber sequencing using chemical cleavage and denaturing gelelectrophoresis (Maxam, A. M. & Gilbert, W. Proc Natl Acad Sci USA 74,560-564 (1977)), pyro-sequencing detection pyrophosphate (PPi) releasedduring the DNA polymerase reaction (Ronaghi, M., Uhlen, M. & Nyren, P.Science 281, 363, 365 (1998)), and sequencing by hybridization (SBH)using oligonucleotides (Lysov, I., Florent'ev, V. L., Khorlin, A. A.,Khrapko, K. R. & Shik, V. V. Dokl Akad Nauk SSSR 303, 1508-1511 (1988);Bains W. & Smith G. C. J. Theor. Biol 135, 303-307 (1988); Drnanac, R.,Labat, I., Brukner, I. & Crkvenjakov, R. Genomics 4, 114-128 (1989);Khrapko, K. R., Lysov, Y., Khorlyn, A. A., Shick, V. V., Florentiev, V.L. & Mirzabekov, A. D. FEBS Lett 256. 118-122 (1989); Pevzner P. A. JBiomol Struct Dyn 7, 63-73 (1989); Southern, E. M., Maskos, U. & Elder,J. K. Genomics 13, 1008-1017 (1992)).

One method of detecting a genotype is to use primers that are specificfor a particular genotype. For example, a primer may be designed toefficiently amplified one genotype but inefficiently or not amplifyanother genotype at all. In an embodiment, the primer may comprise a 3′sequence that is complementary to one genotype (e.g., a genetic diseasegenotype) but not to another genotype (e.g., a normal genotype).

The genotype to be determined may be indicative of a disease such as,for example, the presence of an activated oncogene; the presence of thegene for Huntington's disease or the absence of an anti-oncogene.

The 3′ bases of the primers are especially important in determining thespecificity and efficiency of an RPA reaction. A primer may be designedso that the 3′ base is complementary to one genotype and notcomplementary to another genotype. This will allow efficient RPA of onegenotype and an inefficient RPA (if any) of the second genotype. It isnoted that the method is effective if only one primer of the primer paircan differentiate between different phenotypes (by having differentefficiencies of amplification). In a preferred embodiment, both primersin an RPA reaction can differentiate between different genotypes. Inthis above example, the primers are complementary to one genotype andare not complementary to a second genotype by one base at its 3′ end. Ina preferred embodiment, the primer is not complementary to the secondgenotype by at least one base at its 3′ end. Preferably, the primer isnot complementary to the second genotype by at least 2, 3, 4, 5, 6, 7,8, 9, or 10 bases at its 3′ end. Most preferably, the primer iscompletely non-complementary or cannot hybridize to the second genotypewhile it can hybridize to said first genotype.

In some of the methods discussed, the presence or absence of anamplified product provides the indication of the presence or absence ofa genotype. In these cases, the RPA reaction may be monitored by themethods discussed throughout the specification.

In a preferred embodiment, an RPA reaction for genotyping will amplify asequence regardless of the genotype of the patient. However, thegenotype of a patient will alter a characteristic of the amplifiedsequence. For example, the amplified sequence may be a different size,or sequence for one genotype than for another genotype. In that way, theRPA reaction will contain an internal control to indicate that theamplification reaction was performed successfully. Naturally, a methodof RPA, which includes one or more additional pairs of primers ascontrols for the performance of the RPA reaction, is also envisioned.

In another embodiment, an RPA reaction may be used to determine thepresence or absences of a nucleic acid molecule. The nucleic acidmolecule may be from any organism. For example, the microbialcomposition of a sample may be determined by using a battery of RPAreactions directed to the nucleic acid of different microbes. RPA isespecially useful for the detection of microbes. In one embodiment, thepathogens are selected from viruses, bacteria, parasites, and fungi. Infurther embodiments, the pathogens are viruses selected from influenza,rubella, varicella-zoster, hepatitis A, hepatitis B, other hepatitisviruses, herpes simplex, polio, smallpox, human immunodeficiency virus,vaccinia, rabies, Epstein Barr, retroviruses, and rhinoviruses. Inanother embodiment, the pathogens are bacteria selected from Escherichiacoli, Mycobacterium tuberculosis, Salmonella, Chlamydia andStreptococcus. In yet a further embodiment, the pathogens are parasitesselected from Plasmodium, Trypanosoma, Toxoplasma gondii, andOnchocerca. However, it is not intended that the present invention belimited to the specific genera and/or species listed above.

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EXAMPLES Example 1 Leading Strand Recombinase-Polymerase Amplification(lsRPA)

DNA sequences can be amplified using leading strand synthesis accordingto the Recombinase-Polymerase amplification (RPA) method depicted inFIG. 1.

A reaction is assembled with the following composition:

D-LOOP FORMATION/RESOLUTION COMPONENTS: Component Concentration RecA 20μM Single-stranded oligonucleotide primers 0.25 μM ATP 3 mM RecF 0.1 μMRecO 0.13 μM RecR 0.5 μM Single-stranded Binding protein (SSB) 1 to 10μM DNA polymerase V 5 units

POLYMERASE/HELICASE/RESOLVASE MIX: Component Concentration DNAPolymerase 5 units RuvA 0.5 μM RuvB 0.5 μM RuvC 0.5 μM RecG 10 nM

REACTION BUFFER: Component Concentration MgCl2 2 to 10 mM TrisHCl pH 7.210 mM DTT 0 to 10 mM KCl 0 to 50 mM Deoxyribonucleotide triphosphates0.2 mM Bovine serum albumin (BSA) 0 to 10 μg per ml

The reaction is assembled so that the final concentration satisfies theD-Loop Formation/Resolution Components, Polymerase/Helicase/ResolvaseMix, and Reaction Buffer with the DNA polymerase and/or template addedlast if necessary. For example, a 2× concentrated solution of D-LoopFormation/Resolution Components and of the Polymerase/Helicase/ResolvaseMix may be made in 1× reaction buffer. The reaction may be initiated bymixing an equal volume of each of the two components (each in 1×reaction buffer). Optionally, and as stated above, the DNA polymerase ortemplate (target DNA) may be added last. The reaction is incubated for asufficient time of until the reactants are exhausted. Typical incubationtimes would range from 1 hour, 2 hours, 3 hours, 5 hours, 10 hours orovernight (about 16 hours). Unlike PCR, which requires small volumes forrapid temperature change, there is no limit to the reaction volume ofRPA. Reaction volumes of 25 ul, 50 ul, 100 ul, 1 ml, 10 ml and 100 ml orlarger may be performed in one vessel. Incubation temperature may betypical laboratory temperatures such as 25° C., 30° C. or 37° C.

Prior to the addition of template DNA and/or Polymerase, RecA and SSBwill compete for binding to single-stranded oligonucleotide primers. Inthe presence of a RecR and RecO, RecA is selectively stabilized onto thesingle-stranded primers forming RecA nucleoprotein filaments in acomplex with RecO and RecR. This complex is competent to invadedouble-stranded DNA to form a D-loop at sites homologous to theoligonucleotide primers. Alternatively, RecA, RecO and RecR can bepre-loaded onto oligonucleotide primers prior to the introduction of SSBto the reaction mixture (FIG. 1). The invading strands will be extendedby the polymerase in a 5′ to 3′ direction. As D-loops are formed andsynthesis proceeds displaced single stranded DNA becomes coated withSSB. RecA release from double-stranded DNA can occur via ATP hydrolysisin a 5′ to 3′ direction or as a result of helicase/resolvase orpolymerase activity (FIG. 2A, B). New rounds of invasion/synthesis willcontinuously occur. The third round of strand-invasion/synthesis willrelease discrete products released whose ends correspond to the twofacing primer sites. These fragments will soon become the dominantreaction product and will accumulate to high levels. As each syntheticcomplex processes to the end of the template RecA protein is displacedeither by polymerase activity or by the activity of helicases, such asRuvAB or resolvases, such as RuvC. Once primers, ATP, deoxynucleosidetriphosphates, or any other limiting component is exhausted, thereaction will stop.

The inclusion of temperature-sensitive RecA mutants will allow thecontrolled initiation of DNA synthesis. In such a situation, theinitiation reaction is performed at 25 to 37° C. permitting theformation of D-loops. Elongation reactions are performed at 42° C.,which is non-permissive for RecA mediated double-strand invasion. Thenumber of cycles will determine the amount of reaction product. Extendedelongation phases will permit the amplification of extremely long DNAswithout interference of re-invasion.

Example 2 Nested RPA

The RPA reaction is performed as described in Example 1. A fraction ofone tenth ( 1/10) and one hundredth ( 1/100) of the reaction is removedand used in place of the DNA template in a second round of RPA. lsRPA,leading/lagging RPA, and combinations thereof may be used for nestedRPA.

Example 3 Simultaneous Leading And Lagging Strand Recombinase-PolymeraseAmplification

DNA sequences can be amplified using simultaneous leading and laggingstrand synthesis according to the Recombinase-Polymerase amplification(RPA) method depicted in FIG. 2.

A reaction is assembled with the following composition:

D-LOOP FORMATION/RESOLUTION COMPONENTS Component Concentration RecA 20μM Single-stranded oligonucleotide primers 0.25 μM ATP 3 mM RecF 0.1 μMRecO 0.13 μM RecR 0.5 μM Single-stranded Binding protein (SSB) 1 to 10μM DNA polymerase V 5 units

HELICASE/RESOLVASE MIX Component Concentration RuvA 0.5 μM RuvB 0.5 μMRuvC 0.5 μM RecG 10 nM

PRIMOSOME COMPLEX Component Concentration PriA 20 nM PriB 20 nM DnaT 100nM DnaB 100 nM DnaC 200 nM DnaG 200 nM

DNA POLYMERASE III HOLOENZYME COMPLEX Component Concentration β-Clamp 2μM DnaX Clamp Loader 500 nM Polymerase Core Complex 500 nM

LAGGING STRAND MIX Component Concentration DNA polymerase I 5 units DNAligase 2 units

REACTION BUFFER Component Concentration MgCl₂ 2 to 10 mM TrisHCl pH 7.210 to 20 mM DTT 0 to 10 mM KCl 0 to 50 mM Deoxyribonucleotidetriphosphates 0.2 to 0.4 mM Bovine serum albumin (BSA) 0 to 10 μg per ml

The reaction is assembled so that the final concentration of all thereagents is as listed above. Thus, for example, a 5 fold concentratedsolution of each of the components (D-loop Formation/ResolutionComponents, Helicase/Resolvase Mix, Primosome Complex, DNA PolymeraseIII holoenzyme Complex, Lagging Strand Mix) is made in 1× reactionbuffer. Then, the five solutions are mixed together in equal volumes toinitiate the reaction. The reaction is incubated for a sufficient timeof until the reactants are exhausted. Typical incubation times wouldrange from 1 hour, 2 hours, 3 hours, 5 hours, 10 hours or overnight(about 16 hours). As stated above, there is no limit to the reactionvolume of RPA. Reaction volumes of 25 ul, 50 ul, 100 ul, 1 ml, 10 ml and100 ml or larger may be performed in one vessel. Incubation temperaturemay be typical laboratory temperatures such as 25° C., 30° C. or 37° C.

First, the primosome loads onto the D-loop formed by RecA nucleoproteinfilament invasion. The primosome synthesizes a stretch of RNA primer.Finally, primosome recruits the clamp loader, which recruits both thesliding clamp dimer and the asymmetric DNA polymerase core (FIG. 3A).

Synthesis occurs simultaneously in both the leading and laggingdirections. Eventually lagging strand synthesis stops and the laggingstrand clamp is unloaded. Synthesis of the leading strand continuesuntil a new site of lagging stand synthesis is formed (FIG. 3B).

While leading strand synthesis continues, a new site of lagging standsynthesis is formed. Lagging strand synthesis continues back to theprevious Okazaki fragment where the lagging strand clamp is unloaded(FIG. 3C).

DNA Polymerase I removes the RNA primer, and fills in the gap while DNAligase connects the two Okazaki fragments forming a continuous laggingstrand (FIG. 3D).

1.-72. (canceled)
 73. A composition comprising: i. a polymerase ii. arecombinase agent iii. at least one oligonucleotide primer comprising atleast one modification selected from the group consisting of a modifiedbase, a modified linker backbone, a modified 5′ end, or a modified 3′end iv. a single strand binding protein; and v. ATP, an ATP analogue, oranother nucleoside triphosphate.
 74. The composition of claim 73,wherein the polymerase is derived from a prokaryotic, viral, phage oreukaryotic source.
 75. The composition of claim 74, wherein thepolymerase is a prokaryotic polymerase.
 76. The composition of claim 75,wherein the polymerase is from E. coli.
 77. The composition of claim 76,wherein the polymerase is selected from the group consisting of E. colipolymerase I, E. coli polymerase II, E. coli polymerase III, E. colipolymerase IV and E. coli polymerase V.
 78. The composition of claim 74,wherein the polymerase is a eukaryotic polymerase.
 79. The compositionof claim 78, wherein the eukaryotic polymerase is selected from thegroup consisting of pol-α, pol-β, pol-δ, and pol-ε.
 80. The compositionof claim 73, wherein the recombinase agent is RecA, RadA, RadB or Rad51or a functional analogue or homologue thereof.
 81. The composition ofclaim 80, wherein the recombinase agent is a homologue of RecA obtainedfrom a thermophilic organism, a prokaryote, a vertebrate or a plant. 82.The composition of claim 81, wherein the recombinase agent is ahomologue of RecA obtained from Thermococcus koadkaraensis, Thermotogamatitima, Aquifex pyrophilus, Pyrococcus furiosus, Thermus aquaticus,Pyrobaculum islandicum or Thermus thermophilus, Salmonella typhimurium,Bacillus subtilis, Streptococcus pneumonia, Bacteroides fragilis,Proteus mirabilis Rhizobium meliloti, Pseudomonas aeruginosa,Saccharomyces cerevisiae, Ustilago maydis, Homo sapiens, Xenopus laevisor broccoli.
 83. The composition of claim 80, wherein the recombinaseagent is a peptide comprising residues 193-212 of E. coli RecA
 84. Thecomposition of claim 80, wherein the recombinase agent is a temperaturesensitive mutant.
 85. The composition of claim 73, wherein the at leastone oligonucleotide primer comprises DNA, RNA and/or PNA.
 86. Thecomposition of claim 73, wherein the at least one oligonucleotide primercomprises a modified 5′ end, a modified 3′ end, or both, and wherein themodification comprises a detectable label, a protecting or blockinggroup, a magnetic tag and/or a protein.
 87. The composition of claim 86,wherein the detectable label is selected from the group consisting of anenzyme, an enzyme substrate, a coenzyme, an enzyme inhibitor, afluorescent marker, a quencher, a chromophore, a luminescent marker, aradioisotope (including a radionucleotide) and one member of a bindingpair.
 88. The composition of claim 73, comprising a first and a secondoligonucleotide primer wherein the first oligonucleotide primer, thesecond oligonucleotide primer, or both the first and the secondoligonucleotide primers comprise at least one modification selected fromthe group consisting of a modified base, a modified linker backbone, amodified 5′ end, or a modified 3′ end.
 89. The composition of claim 88,wherein the first oligonucleotide primer is at least partiallycomplementary to a first portion of a first strand of a double strandedtarget nucleic acid and the second oligonucleotide primer is at leastpartially complementary to a portion of a second strand of the doublestranded target nucleic acid, and wherein the 3′ ends of the first andsecond oligonucleotide primers are pointed towards each other whenhybridised to the target nucleic target.
 90. The composition of claim89, further comprising one or more additional oligonucleotide primersdesigned to be internal to the first and second oligonucleotide primers.91. The composition of claim 90, wherein at least one of the additionaloligonucleotide primers comprises at least one modification selectedfrom the group consisting of a modified base, a modified linkerbackbone, a modified 5′ end, or a modified 3′ end.
 92. The compositionof claim 73, comprising three oligonucleotide primers wherein any one,any two, or all three of the oligonucleotide primers comprises amodification selected from the group consisting of a modified base, amodified linker backbone, a modified 5′ end, or a modified 3′ end. 93.The composition of claim 92, wherein one oligonucleotide primercomprises a 3′ blocking group.
 94. The composition of claim 73, whereinthe at least one oligonucleotide primer is partially single stranded andpartially double stranded.
 95. The composition of claim 94, wherein theat least one oligonucleotide primer comprises a longer invading-strandwhich possesses a specific 3′ primer sequence and a shorter non-invadingstrand, which is complementary to the 5′ end of the longer invadingstrand.
 96. The composition of claim 95, wherein the invading strand,the non-invading strand, or both the invading strand and thenon-invading strand are labeled with a detectable label.
 97. Thecomposition of claim 73, wherein the ATP, an ATP analogue, or anothernucleoside triphosphate is ATP and wherein the composition furthercomprises RecO, RecR and/or RecF
 98. The composition of claim 73,wherein the ATP analogue is ATPγS, dATP, ddATP or UTP.
 99. Thecomposition of claim 98, wherein the ATP analogue is ATPγS and whereinthe composition further comprises a helicase or PolV.
 100. Thecomposition of claim 73, further comprising one or more accessory agentsselected from the group consisting of topoisomerase, resolvase, asliding clamp, DNA Polymerase III holoenzyme complex, RuvA, RuvB, RuvC,RecG, PriA, PriB, DnaT, DnaC, DnaG and DNA ligase.
 101. The compositionof claim 73, further comprising dATP, dTTP, dCTP, dGTP or derivativesand analogues thereof.